Split G-Quadruplexes for Capture and Detection of Nucleic Acids

ABSTRACT

Methods of using split G-quadruplexes associated with functional tags for associating said tags to target nucleic acids. Methods include use of split G-quadruplexes associated with detection tags for the detection of target nucleic acids, and use of split G-quadruplexes associated with capture tags for detection or capture of target nucleic acids.

The invention relates to methods of associating functional tags totarget nucleic acids, and uses thereof, including the detection oftarget nucleic acids, and the capture of target nucleic acids.

BACKGROUND OF THE INVENTION

G-quadruplexes are structures formed in nucleic acids by sequences thatare rich in guanine. Four guanine bases can associate through Hoogsteenhydrogen bonding to form a square planar structure called a guaninetetrad, and two or more guanine tetrads can stack on one another to forma G-quadruplex. The quadruplex structure is further stabilized by thepresence of a cation, which sits in a central channel between each pairof tetrads. They can be formed of DNA or RNA (or other nucleic acids);and they typically form from one strand (intramolecular), two strands(bimolecular), three strands (trimolecular), or four stands(tetramolecular) of nucleic acid. G-quadruplexes with 4 G-rich sequencesaligned in the same 5′-to-3′ direction are termed parallel;G-quadruplexes with 2 G-rich sequences aligned 5′-to-3′ and 2 G-richsequences aligned 3′-to-5′ direction are termed anti-parallel; and Gquadruplexes with either 3 G-rich sequences aligned 5′-to-3′ and 1G-rich sequence aligned 3′-to-5′, or 1 G-rich sequence aligned 5′-to-3′and 3 G-rich sequences aligned 3′-to-5′, are termed mixed or hybrids.

Bases intervening the G-rich sequences are required for proper foldingof the G-quadruplex, especially for bimolecular and intramolecularstructures. Upon G-quadruplex formation, these bases reside in loopregions, and G-quadruplexes of different topologies have loops indifferent configurations. For example, quadruplexes in a paralleltopology will have loops in a propeller configuration (positioned to theside of the quadruplex), whereas quadruplexes in an anti-paralleltopology will have loops in a lateral configuration (joining adjacentG-rich sequences), or in both lateral configuration and diagonalconfiguration (joining diagonally opposite G-rich sequences). Further,studies have developed the consensus sequence G₃₊N₁₋₇₊G₃₊N₁₋₇₊G₃₊N₁₋₇₊G₃(where N is any base including guanine) to identify putativeG-quadruplexes, with G₃ representing the G-rich sequences, and N₁₋₇representing the intervening bases in the loop regions. Examples ofpublished and theoretical G-quadruplexes are found on the G4RNA Database(http://scottgroup.med.usherbrooke.ca) and GregList (G-quadruplexRegulated Genes List) (http://tubic.tju.edu.cn/greglist), respectively.

G-quadruplex sequence is present in the genomes of a variety oforganisms. In humans, genome-wide surveys have identified >376,000Putative Quadruplex Sequences, although not all of these probably formin vivo. Some sequences are found in human telomeres with the DNA repeatd(GGTTAG)_(n). The formation of quadruplexes in telomeres has been shownto decrease the activity of the enzyme telomerase, which is responsiblefor maintaining the length of telomeres, and is involved in around 85%of all cancers. Other sequences are found in promoter regions of genes,including the proto-oncogenes c-myc, k-ras, c-kit, Bcl-2, and VEGF.

In addition to cations, other molecules have been identified thatinfluence formation of or bind to G-quadruplexes. Some molecules induceformation of G-quadruplexes, including the DNA binding protein RAPT, thecrowding agent polyethylene glycol, and the ionic liquid guanidiniumtris(pentafluoroethyl)trifluorophosphate (Gua-IL). Other molecules arecapable of binding G-quadruplexes, including the helicase BLM, the DNAbinding protein RAP1, the engineered zinc finger protein Gq1, theG-quadruplex-specific antibody 1H6, and the small molecules hemin, NMM,TMPyP4, and telomestatin. Interestingly, a subset of these moleculesalso stabilizes formation of G-quadruplexes (ex. Gua-IL, TMPyP4, andtelomestatin). The G-quadruplex Ligands Database (http://www.g41db.org)lists hundreds of molecules that influence or bind G-quadruplexes.

Catalytic G-Quadruplexes

G-quadruplexes have been isolated from random DNA libraries usingaptamer selection methodology SELEX and the molecule NMM—a transitionstate analog of heme, an enzyme cofactor found in peroxidases and otherenzymes. G-quadruplexes bind NMM with micromolar affinity (viaend-stacking), and interestingly, bind hemin (an oxidized form of heme)with micromolar affinity too. With addition of oxidizing agent H₂O₂, theG-quarduplex-hemin complex is capable of oxidizing a variety ofsubstrates, including colorimetric and chromogenic substrates (ex. DAB,ABTS)—and chemiluminescent substrates (ex. luminol)—used in peroxidaseassays. The G-quadruplex-hemin complex is approximately two orders ofmagnitude more reactive than hemin alone in catalyzing peroxidasereactions.

Hence, G-quadruplexes are considered to be DNA enzymes. Studies haveshown that G-quaduplex-hemin complexes are less active than horseradishperoxidase (HRP) but more active than the enzyme catalase.Interestingly, the G-quadruplex-hemin complex displays a broader rangeof substrate specificity than HRP, and a higher rate ofself-inactivation than HRP—likely because of a more exposed active site.Some studies have shown G-quadruplex activity dependent on ions,buffers, pH, and surfactants—as well as activity enhancement agents suchas adenosine triphosphate and spermidine. Other studies have shownG-quadruplex activity dependent on loop size, flanking sequence, andtopology. G-quadruplex-hemin complexes have many advantages incomparison to HRP; however, weaker peroxidase activity and higherinactivation rate have hindered G-quadruplex use as HRP replacements.

Split G-Quadruplexes

Split G-quadruplexes are engineered G-quadruplexes that are used todetect nucleic acids via their inherent catalytic activity. Thesemolecules were first designed by dividing the G-quadruplex sequence intoan upstream sequence and a downstream sequence, and attachingtarget-binding arms to the upstream and downstream sequences. Hence,split G-quadruplexes comprise two oligonucleotide strands—each with apartial G-quadruplex sequence and a target-binding arm. Thetarget-binding arms are single stranded, and designed to bind singlestranded target nucleic acid, for example, designed with sequencecomplementary to the target nucleic acid, and thus capable of bindingsaid nucleic acid. Accordingly, in the presence of the target, the splitG-quadruplex binds and its G-quadruplex assembles and becomes competentto catalyze its peroxidase reaction.

The use of split G-quadruplexes with peroxidase activity to detectnucleic acids is interesting, especially with molecules demonstratinghigh binding specificity (using short target binding arms). However,studies have observed low target sensitivity (ex. 10 nM-to-1 mM usingcolorimetric substrates) in comparison to HRP assays (ex. 0.1 pM-to-100pM). The low target sensitivity probably reflects the aforementionedlimitations of (i) weaker peroxidase activity and (ii) higherinactivation rate in comparison to HRP. As with G-quadruplexes, theselimitations have similarly hindered use of split G-quadruplexes asnucleic acid detection agents.

SUMMARY OF THE INVENTION

The present invention describes methods and reagents for associatingtags to nucleic acids, the method comprising associating a tag to asplit G-quadruplex, and binding a split G-quadruplex to a nucleic acid.In some aspects, the tag is a capture tag, which can be used to capturea split G-quadruplex, and accordingly, capture a nucleic acid bound bythe split G-quadruplex. In other aspects, the tag is a detection tag,which can be used to detect a split G-quadruplex, and accordingly,detect a nucleic acid bound by the split G-quadruplex.

Tags are nucleic acid modifications that impart characteristic featuresto the nucleic acid, such as the ability to be captured, detected,targeted, or crosslinked. Tags are known in the art, commonly sold byoligonucleotide manufacturers, and can be bound or incorporated intonucleic acids, such as attachment chemistries, fluorophores, detectableenzymes, detectable particles, and nucleotide analogs.

In some aspects, the disclosure provides a kit for capturing ordetecting nucleic acids comprising a split G-quadruplex and anassociated tag used to capture or detect said split G-quadruplex. Inother aspects, the disclosure provides an apparatus for capturing ordetecting nucleic acids comprising a split G-quadruplex and anassociated tag used to capture or detect said split G-quadruplex.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an illustration of six embodiments of the disclosed inventionfor the capture or detection of target nucleic acids (dashed line)utilizing split G-quadruplexes associated with capture tags (C) ordetection tags (D). In the upper box, an antibody (Ab), a splitG-quadruplex (SQ), and a split-G-quadruplexes with capture arms (SQB)are illustrated. Some embodiments feature a capture nucleic acid (solidline). The capture tag of the nucleic acid used to bind the solidsurface (large black rectangle) is not illustrated.

DESCRIPTION OF THE INVENTION

The present invention describes a surprisingly stable interactionbetween split G-quadruplexes and target nucleic acids. The high bindingaffinity of split G-quadruplexes (for target nucleic acids) does notrequire peroxidase activity—and likely reflects formation of theG-quadruplex upon target binding, which physically links the two targetbinding arms of the split G-quadruplex. The combination of high bindingaffinity and high binding specificity are features shared withantibodies, which are used to associate (or bridge) tags to antigens.Accordingly, the present invention discloses the use of splitG-quadruplexes to associate tags to nucleic acids, for example, inmethodologies to capture nucleic acids, and methodologies to detectnucleic acids. The present invention also discloses the use of splitG-quadruplexes (in place of antibodies) in several antibodymethodologies adapted for nucleic acids, including purification,precipitation, targeting, crosslinking, and modification of nucleicacids—as well as kits and apparatuses based on said methodologies.

Split G-Quadruplexes

Split G-quadruplexes are engineered G-quadruplexes designed to bindtarget sequences in target nucleic acids, and upon binding, assembleinto G-quadruplexes. These molecules can comprise two, three, or fourstrands of nucleic acid, with (i) each strand containing a partialsequence of a G-quadruplex, and (ii) two or more strands withsingle-stranded target-binding arms, each capable of binding part of asingle-stranded target sequence of the target nucleic acid. In oneembodiment, target-binding arms are designed with sequence capable ofbinding the target sequence, for example, by hybridization. In anotherembodiment, target-binding arms are designed with sequence complementaryto the target sequence, and thus are capable of binding the targetsequence by hybridization. In a preferred embodiment, the sum of thepartial sequences of the split G-quadruplex is a G-quadruplex, and thesum of the target-binding arm sequences is a sequence complementary tothe target sequence.

A split G-quadruplex of x strands (where x=2, 3, or 4) can be designedby (i) dividing the G-quadruplex sequence into x partial sequences (ex.for x=2, divided into an upstream and downstream sequence, ex.containing 1, 2, or 3 G-rich sequences); (ii) determining thecomplementary sequence of the target sequence, dividing thecomplementary sequence into y partial sequences (where y=2, 3, or 4);and (iii) placing a partial complementary target sequence on each strandof the split G-quadruplex. The partial complementary target sequences(herein called the target binding arms) can be placed on the 5′-end,3′-end, or in an internal position of the partial G-quadruplex sequence.For example, a split G-quadruplex of two strands can have a first strandwith either (i) the 5′-end of the target binding arm (with upstreamcomplementary target sequence) attached to the 3′-end of the upstreamG-quadruplex sequence, or preferentially (ii) the 3′-end of the targetbinding arm (with upstream complementary target sequence) attached tothe 5′-end of the upstream G-quadruplex sequence; and a second strandwith either (iii) the 5′-end of the downstream G-quadruplex sequenceattached to the 3′-end of the target binding arm (with downstreamcomplementary target sequence), or preferentially (iv) the 3′-end of thedownstream G-quadruplex sequence attached to the 5′-end of the targetbinding arm (with downstream complementary target sequence). In anotherembodiment, a split G-quadruplex strand is attached to two or moretarget binding arms. In a preferred embodiment, the target-binding armsare attached to the partial G-quadruplex sequences via a linker orspacer—such as Phosphoramidite C3, Hexanediol, and 1′,2′-Dideoxyribose,and preferentially via the spacers Triethylene Glycol orHexa-Ethyleneglycol.

Split G-quadruplex strands can be designed with different combinationsof partial G-quadruplex sequences. For example, a split G-quadruplex oftwo strands can have a first strand with 3 G-rich sequences and 2 loopsof a G-quadruplex, and a second strand with 1 G-rich sequence of aG-quadruplex. In a preferred embodiment, the first strand and secondstrands each comprise 2 G-rich sequences and 1 loop of a G-quadruplex.In another embodiment, the first strand comprises the G-quadruplexsequence G₃₊N₁₋₇₊G₃₊N₁₋₇₊G₃, and the second strand comprises theG-quadruplex sequence G₃, wherein N is any base including guanine. In apreferred embodiment, the first and second strand sequences eachcomprise the G-quadruplex sequence G₃₊N₁₋₇₊G₃, wherein N is any baseincluding guanine. In another preferred embodiment, the first strandcomprises the G-quadruplex sequence GGGTAGGG, and the second strandcomprises the G-quadruplex sequence GGGTTGGG. And split G-quadruplexesof three and fours strands can be designed by dividing (splitting) theabove G-rich sequences, such as one 2 G-rich and two 1 G-rich strandsfor a three strand G-quadruplex, and four 1 G-rich strands for a fourstrand quadruplex.

A target sequence can be a single continuous sequence in the targetnucleic acid. Accordingly, in one embodiment, the target binding arms ofthe split G-quadruplex are designed to bind two flanking regions (parts)of said continuous target sequence. A target sequence can also be two ormore physically separate sequences (ex. parts, separated by non-targetsequence) in the target nucleic acid. Accordingly, in anotherembodiment, the target-binding arms of the split G-quadruplex aredesigned to bind two or more physically separate sequences of the targetsequence. In another embodiment, one or more target binding arms aremade short in length, so in the chosen conditions and temperature forbinding, the split G-quadruplex hybridizes to perfect target sequencesand not alternative sequences, including sequences with nucleotidesubstitutions, such as single nucleotide polymorphisms (SNPs). Forexample, the short length can be selected by (1) choosing a temperaturefor operation of the split G-quadruplex (ex. 25° C.), and (2) designinga pair of target binding arms, with one arm having a melting temperature(Tm) equal or greater than the operation temperature (ex. 40° C.), andone arm having a Tm equal of less than the operation temperature (ex.20° C.), wherein a nucleotide substitution in the target significantlylowers the Tm (ex. <0° C.). Such a design permits the use of the splitG-quadruplexes to capture or detect perfect target sequences, oralternatively, capture or detect sequences with nucleotidesubstitutions, including SNPs.

In one embodiment, split G-quadruplexes are used with molecules thatinfluence the formation of G-quadruplexes, or bind to G-quadruplexes. Ina preferred embodiment, split G-quadruplexes are used with moleculesthat induce the formation of G-quadruplexes, such as cations (ex. Na⁺,K⁺, NH₄ ⁺), the DNA binding protein RAPT, the crowding agentpolyethylene glycol, and the ionic liquid guanidiniumtris(pentafluoroethyl)trifluorophosphate (Gua-IL). In another preferredembodiment, split G-quadruplexes are used with molecules that stabilizethe formation of G-quadruplexes, such as Gua-IL, TMPyP4, andtelomestatin. And in another preferred embodiment, split G-quadruplexesare used with molecules that promote the catalytic activity ofG-quadruplexes, such as ATP.

Melting temperature studies have found G-quadruplexes with paralleltopologies to be more stable than G-quadruplexes with anti-paralleltopologies. In the present invention, split G-quadruplexes with greaterstability—ex. with parallel topology—are expected to bind targets morestably, and can be used in methods requiring improved target binding.Similarly, split G-quadruplexes with weaker stability—ex. withanti-parallel topology—are expected to bind targets more weakly, and canbe used in methods requiring reduced target binding (ex. to reducebackground or improve specificity). G-quadruplexes can be designed ortreated (ex. with chemicals) to assume different topologies, such as aparallel topology, an anti-parallel topology, or a hybrid topology. Forexample, G-quadruplexes designed with short loops favor paralleltopologies, and G-quadruplexes with long loops favor anti-paralleltopologies. For example, G-quadruplexes treated with K⁺ favor paralleltopologies, and G-quadruplexes treated with Na⁺ favor anti-paralleltopologies. In the present invention, split G-quadruplexes can besimilarly designed or treated to favor parallel topologies or favoranti-parallel topologies.

Tags

Tags are nucleic acid modifications that can be associated—ex. bound orincorporated—to nucleic acids, and similarly, can be associated—ex.bound or incorporated—to split G-quadruplexes. Tags are functional, andtheir association to a nucleic acid imparts their function to thenucleic acid. For example, a tag that can be detected (ex. a detectiontag), bound or incorporated to a nucleic acid, permits said nucleic acidto be detected. For example, a tag that can be captured (ex. a capturetag), bound or incorporated to a nucleic acid, permits said nucleic acidto be captured. For example, a tag that can be targeted (ex. a targettag), bound or incorporated to a nucleic acid, permits said nucleic acidto be targeted. For example, a tag that can be crosslinked (ex. acrosslinked tag), bound or incorporated to a nucleic acid, permits saidnucleic acid to be crosslinked.

Tags are usually associated to nucleic acids by binding or incorporationto the nucleic acid (ex. during nucleic acid synthesis). Tags andnucleic acid modifications are known in the art, and many are availablefrom oligonucleotide manufacturers. Examples of detection tags includefluorophores, quenchers, phosphoylation, detectable enzymes (horseradishperoxidase), dyes, reactants (ex. acrydite), detectable particles, andnucleotide analogs (ex. fluorescent, radiolabeled); and includeacrydite, Cyanine dyes, 6-FAM, Fluorescein-dT, HEX, JOE, Lightcycler640, ROX, SYBR Green, TAMRA, TET, Texas Red-X, Alexa Fluor dyes,Rhodamine dyes, WellRED dyes, Black Hole quenchers, DABCYL, and2-aminopurine. Examples of capture tags include attachment chemistries,binding chemistries, phosphorylation, antibody antigens, antibodies,nucleotide analogs (ex. that are antibody antigens), and nucleotidesequences (ex. that hybridize to other nucleic acids); and includeadenylation, alkyne modifiers (ex. click reaction), amino modifiers,avidin, azide, biotin, cholesterol, digoxigenin (DIG), 2,4-dinitrophenol(DNP), and thiol modifiers. Examples of target tags include cholesteroland phosphorylation. Examples of crosslink tags include5-bromo-deoxyuridine. Other tags, such as nucleotide analogs (whichinclude modified nucleotides, ex. nucleotides with modifiednucleobases), have members with functions similar to the aforementionedtags, such as 2-aminopurine (detection) and 5-bromo-2′-deoxyuridine(BrdU) (crosslinking).

Detection tags are nucleic acid modifications that can be detected, forexample, by senses (ex. visually), or by use of assays or equipment (ex.measuring the presence, amount, or functional activity of the detectiontag). Commonly used detection tags are fluorophores and quenchers, whichcan be detected by fluorometer or microscope. Other detection tags thatcan be used include phosphorylation and nucleotide analogs (ex.radiolabeled and detected by scintillation counter or autoradioagraphy(ex. film)), detectable enzymes (ex. horseradish peroxidase), detectableparticles (ex. colloidal gold and colored latex), and BrdU (ex.crosslinking the labeled nucleic acid (ex. the split G-quadruplex) withthe target nucleic acid, and then measuring the presence of the twocrosslinked nucleic acids by electrophoresis or chromatography).

Capture tags are nucleic acid modifications that bind, or are capable ofbinding, to specific molecules—herein called capture targets. Somecapture tags rely on high-affinity non-covalent bonds for capture targetbinding, such as biotin (binding to avidin) and digoxigenin and2,4-dinitrophenol (binding to anti-DIG and anti-DNP antibodies,respectively). Other capture tags rely on covalent bonds for capturetarget binding, which often require chemical treatment to activatereactive groups on the capture tag (or capture target), such as aminomodifiers, alkyne modifiers, and thiol modifiers. Examples of capturetargets include nucleic acids (including the herein capture nucleicacids), molecules than can be detected (ex. dyes and enzymes), moleculescapable of binding other molecules (ex. antigens and antibodies), andsolid surfaces.

Target tags such as cholesterol and phosphorylation can be used tofacilitate nucleic acid uptake into cells. Target tags can be similarlyincorporated into a split G-quadruplex, for example, to facilitate itsuptake into cells, and to facilitate the uptake of target nucleic acidsbound by the split G-quadruplex. Such split G-quadruplexes can also beassociated with DNA regulatory molecules—ex. enzymes, nucleases,transcription factors, enzyme inhibitors, enzymes subtrates, enzymescatalysts, etc.—in order to target said molecules to specific targetnucleic acids within cells, for example, for use in gene regulation,protein expression via RNA regulation, or anti-viral or anti-bacterialtherapy. Crosslink tags such as BrdU are used to crosslink targetnucleic acids to other nucleic acids or proteins. Crosslink tags cansimilarly be incorporated into a split G-quadruplex in order tocrosslink it to other nucleic acids, or to other proteins, which thencan be associated to a target nucleic acid by binding said splitG-quadruplex to the target nucleic acid.

Split G-quadruplexes can contain one or more tags—bound or incorporatedin one, two, three, or four of its strands. The tags can be placed onthe 5′-end, 3′-end, or the middle of a strand, and different tags can beplaced on one strand or different strands of the split G-quadruplex. Inone embodiment, tags are placed distant from the partial G-quadruplex,for example, one strand can have a tag bound or incorporated to the5′-end of a target-binding arm, and the 3′-end of the target-binding armattached to the 5′-end of the partial G-quadruplex sequence. Such aconfiguration may be desired if the capture tag—or capture target (ex.solid surface) attached to the capture tag—can interfere with theassembly of the split G-quadruplex. In another embodiment, tags areplaced proximal to the partial G-quadruplex, for example, one strand canhave the 3′-end of the target-binding arm attached to the 5′-end of thepartial G-quadruplex sequence, and the 3′-end of the partialG-quadruplex sequence bound or incorporated with the tag. Such aconfiguration may be desired if multiple capture tags (ex. biotins) canbind a single target molecule (ex. avidin), which can result in multipleG-quadruplex strands being captured next to one another, and assemblingartificially into a G-quadruplex (without target binding arm binding tothe target nucleic acid). Placing the capture tag proximal to thepartial G-quadruplex effectively locates said G-quadruplex sequence to asingle binding site on the capture target, and reduces the probabilitythat said sequence can interact with other G-quadruplexes bound to otherbinding sites on the same or neighboring capture targets. Additionalbinding sites can be blocked by using capture tags that can bindmultiple binding sites on the same or neighboring capture targets, forexample, a commercially available capture tags with two biotins(attached by long linkers).

Capture Arms

Additional target-binding arms—capable of binding other nucleic acidsequences—can be added to split G-quadruplexes to capture other targetnucleic acids. These additional arms function as sequence-dependentcapture tags, capable of binding nucleic acids as their capture targets,and can be designed similarly to, or different from, the aforementionedtarget-binding arms. Accordingly, herein, different nomenclature isutilized, where these additional target-binding arms are called capturearms, which bind target sequences called capture sequences, that arepresent in target nucleic acids called capture nucleic acids. In oneembodiment, capture arms are designed similarly to target-binding arms,wherein each strand of the split G-quadruplex contains a single-strandedcapture arm, which is capable of binding part of the single-strandedcapture sequence of the capture nucleic acid. In a preferred embodiment,one or more capture arms are made short in length, so in the chosenconditions and temperature for binding, the split G-quadruplexhybridizes to perfect capture sequences and not alternative sequences,including sequences with nucleotide substitutions, such as SNPs. Inanother embodiment, capture arms are designed differently totarget-binding arms, wherein one strand of the split G-quadruplexcontains a capture arm, which is capable of binding the entire capturesequence of the capture nucleic acid. And in another embodiment, the sumof the capture arm sequences is a sequence complementary to the capturesequence.

A split G-quadruplex of n strands (where n=2, 3, or 4) and x capturearms (where x=1, 2, 3, or 4, and x≤n) can be designed by (i) dividingthe G-quadruplex sequence into n partial sequences (ex. for n=2, dividedinto an upstream and downstream sequence, ex. containing 1, 2, or 3G-rich sequences), and placing a partial sequence on each strand of thesplit G-quadruplex; (ii) determining the complementary sequence of thetarget sequence, dividing the complementary sequence into n partialsequences (ex. for n=2, divided into an upstream and downstreamsequence), and placing a partial complementary target sequence on eachstrand of the split G-quadruplex; and (iii) determining thecomplementary sequence of the capture sequence, dividing thecomplementary sequence into x partial sequences (ex. for x=2, dividedinto an upstream and downstream sequence), and placing a partialcomplementary capture sequence on one or more strands of the splitG-quadruplex. For example, a split G-quadruplex of two strands can havea first strand with the 3′-end of the target binding arm (with upstreamcomplementary target sequence) attached to the 5′-end of the upstreamG-quadruplex sequence, and the 3′-end of the upstream G-quadruplexsequence attached to the 5′-end of the capture arm (with downstreamcomplementary capture sequence); and the 3′-end of the capture arm (withupstream complementary capture sequence) attached to the 5′-end of thedownstream G-quadruplex sequence, and 3′-end of the downstreamG-quadruplex sequence attached to the 5′-end of the target-binding arm(with downstream complementary target sequence). In a preferredembodiment, the target-binding arms and the capture arms are attached tothe partial G-quadruplex sequences via a linker or spacer—such asPhosphoramidite C3, Hexanediol, and 1′,2′-Dideoxyribose, andpreferentially via the spacers Triethylene Glycol orHexa-Ethyleneglycol.

Capture nucleic acids can be bound or incorporated with functional tags;for example, detection tags, capture tags, target tags, or crosslinkedtags. Accordingly, capture nucleic acids can be used to associatefunctional tags to target nucleic acids; for example (i) a functionaltag is associated to a capture nucleic acid, (ii) said capture nucleicacid is bound to the capture arms of a split G-quadruplex, and (iii) thetarget binding arms of said G-quadruplex is bound to a target nucleicacid. Such methods permit the use of capture nucleic acids with splitG-quadruplexes to detect, capture, target, or crosslink target nucleicacids. For example, methods are described below for the detection oftarget nucleic acids using a capture nucleic acid with a detection tag,and split G-quadruplexes. For example, methods are described below forthe capture of target nucleic acids onto a solid surface using capturenucleic acids with capture tags, and split G-quadruplexes.

Methods to Detect Nucleic Acids

In one embodiment, target nucleic acids are detected by (i) associatinga detection tag to a split G-quadruplex, (ii) binding said splitG-quadruplex to the target nucleic acid, and (iii) detecting thedetection tag with a method known in the art. Examples of detection tagsthat can be used include fluorophores, quenchers, phosphoylation, andnucleotide analogs. In another embodiment, target nucleic acids aredetected by (i) binding a split G-quadruplex to a target nucleic acid,(ii) associating a detection tag to the split G-quadruplex, and (iii)detecting the detection tag with a method known in the art. Examples ofdetection tags that can be used include phosphorylation and nucleotideanalogs. In a preferred embodiment, the target nucleic acid is bound toa solid surface—either (i) before the target nucleic acid is bound tothe split G-quadruplex; or (ii) after the target nucleic acid is boundto the split G-quadruplex, but before detection of the detectiontag—permitting washing of said target nucleic acid and removal ofunbound detection tags before detection (of bound detection tags). Anillustration of a target nucleic acid associated to a detection tagusing said methods and a solid surface (i.e. preferred embodiment) isshown in FIG. 1 Embodiment 1.

A second approach to detect target nucleic acids uses capture tags tobind capture targets that can be detected, or capture targets that canbind detectable molecules. Examples of detectable capture targets (anddetectable molecules) include detection tags and immunoassay labels, anddetectable enzymes, fluorophores, detectable particles, and radiolabeledmolecules. Examples of detectable enzymes include enzymes that catalyzechromogenic or chemiluminescent reactions, such as alkaline phosphatase(AP), horseradish peroxidase (HRP), beta-galactosidase (b-gal), andluciferase (LUC), and DNA enzymes such as Catalytic G-Quadruplexes.Examples of detectable fluorophores include fluorescein isothiocyanate(FITC) and tetramethylrhodamine (TRITC). Examples of detectableparticles include colloidal gold, colored or fluorescent latex, andparamagnetic latex particles. And examples of detectable radiolabeledmolecules include antibodies and antigens labeled with 125-I or 3-H.

In one embodiment, target nucleic acids are detected using capture tagsand capture targets by (i) associating a capture tag to a splitG-quadruplex, (ii) binding said split G-quadruplex to a target nucleicacid, (iii) binding a detectable capture target to said capture tag, and(iv) detecting the detectable capture target with a method known in theart. In another embodiment, target nucleic acids are detected usingcapture tags and capture targets by (i) associating a capture tag to asplit G-quadruplex, (ii) binding a detectable capture target to saidcapture tag, (iii) binding said split G-quadruplex to a target nucleicacid, and (iv) detecting the detectable capture target with a methodknown in the art. And in another embodiment, target nucleic acids aredetected using capture tags and capture targets by (i) binding a splitG-quadruplex to a target nucleic acid, (ii) associating a capture tag tosaid split G-quadruplex, (iii) binding a detectable capture target tosaid capture tag, and (iv) detecting the capture target with a methodknown in the art. In these methods, capture targets that can be detectedcan be utilized, or capture targets capable of binding detectablemolecules can be utilized. Methods of binding capture tags to capturetargets, and binding capture targets to detectable molecules, are knownin the art. In a preferred embodiment, the target nucleic acid is boundto a solid surface—either (i) before the target nucleic acid is bound tothe split G-quadruplex; or (ii) after the target nucleic acid is boundto the split G-quadruplex, but before detection—permitting washing ofsaid target nucleic acid and removal of unbound detectable capturetargets or molecules before detection. An illustration of a targetnucleic acid associated to a detectable capture target using saidmethods and a detectable molecule and a solid surface (i.e. preferredembodiment)—is shown in FIG. 1 Embodiment 2.

In another embodiment, detection methods can be improved if combinedwith secondary methods that (i) amplify the signal of detection tags,detectable capture targets, or detectable molecules; or (ii) captureadditional detection tags, detectable capture targets, or detectablemolecules. For example, methods to amplify signals include methods toimprove the stability of detection tags, detectable capture targets, ordetectable molecules (ex. addition of dextran to HRP); and methods toimprove the catalytic activity of detection tags, detectable capturetargets, or detectable molecules (ex. addition of PEG to prevent HRPinactivation). For example, methods to capture (or cascade) additionaldetection tags, detectable capture targets, or detectable moleculesinclude (i) tyramide signal amplification (TSA), (ii)avidin-biotinylated enzyme complexes (ABC), and (iii) branched-DNAassays (bDNA). In one embodiment, the capture arms of a splitG-quadruplex are used to capture (or cascade) additional detection tags,detectable capture targets, or detectable molecules—for example, bybinding capture nucleic acids that (i) are associated with detectiontags or detectable capture tags, or (ii) are capable of bindingmolecules that can bind or cascade with detectable molecules. In anotherembodiment, the capture arms of a split G-quadruplex are used to capture(or cascade) additional split G-quadruplexes, that optionally havedetection tags or detectable capture tags (ex. additional capture armscapable of binding additional capture nucleic acids or splitG-quadruplexes).

Methods to Capture Nucleic Acids

Methods of using capture tags to bind capture targets are known in theart, and are used to bind nucleic acids—associated with capture tags—tocapture targets, including solid surfaces. Nucleic acids bound to solidsurfaces are used in several methodologies, including nucleic acidprecipitation, nucleic acid purification, branched DNA assays, solidphase PCR amplification, and solid phase bridge amplification (for NGSsequencing). These methodologies can be grouped into two capturemethods, where the first group of capture methods—nucleic acidprecipitation and nucleic acid purification—generally use a targetnucleic acid associated to a capture tag, which is capable of binding asolid surface; and the second group of capture methods—branched DNA,solid phase PCR, and solid phase bridge amplification—generally use anon-target nucleic acid (which is capable of hybridizing to the targetnucleic acid, ex. a primer), which is associated to a capture tag, andthus capable of binding a solid surface. In one embodiment of thepresent invention, split G-quadruplexes associated to capture tags, thatare capable of binding both target nucleic acid (via target-bindingarms) and a solid surface (via a capture tag), can be used to bindtarget nucleic acids to solid surfaces. Accordingly, in theaforementioned methodologies using nucleic acids bound to solidsurfaces, a split G-quadruplex associated to a capture tag plus anucleic acid can substitute the target nucleic acid associated to acapture tag (in the first group) and the non-target nucleic acidassociated to a capture tag plus a nucleic acid (in the second group) ofthe aforementioned methodologies.

In one embodiment, a split G-quadruplex associated to a capture tag canbe used to bind a target nucleic acid to a solid surface by (i)associating a capture tag to the first strand of a split G-quadruplex,(ii) binding said first strand and the second strand of the splitG-quadruplex to a target nucleic acid, and (iii) bind said capture tagto a solid surface. In a second embodiment, a split G-quadruplexassociated to a capture tag can be used to bind a target nucleic acid toa solid surface by (i) associating a capture tag to the first strand ofa split G-quadruplex, (ii) binding said capture tag to a solid surface,and (iii) binding the second strand of the split G-quadruplex and thetarget nucleic acid to the surface bound first strand of the splitG-quadruplex. An illustration of a target nucleic acid associated to acapture tag and solid surface using said methods—is shown in FIG. 1Embodiment 4. The illustration also shows an optional detection tag (D)on the second strand of the split G-quadruplex, which permits detectionof the captured target nucleic acid, preferentially using an added washstep before the detection step to remove unbound detection tags. Inanother embodiment, a split G-quadruplex associated to a capture tag canbe used to bind target nucleic acids to a solid surface, by (i)associating capture tags to the first and second strands of a splitG-quadruplex, (ii) binding said first and second strands of the splitG-quadruplex to a target nucleic acid, and (iii) binding said capturetag to a solid surface.

Embodiments for binding nucleic acids to solid surfaces are useful forthe first group of capture methodologies (including nucleic acidprecipitation and purification), and are useful for the second group ofcapture methodologies (including solid phase PCR and bridgeamplification) when combined with template-directed nucleic acidsynthesis—such as enzymatic methods for DNA synthesis, DNAamplification, DNA transcription, and RNA synthesis. Methods oftemplate-directed nucleic acid synthesis are known in the art, and canbe classified in three groups, requiring (i) a primer (or 3′-OHterminus) for initiation, (ii) a promoter sequence for initiation, or(iii) neither primer or promoter for initiation. These requirements forprimers or promoters can be accommodated by the split G-quadruplex—withor without association to a capture tag—for example, by using the 3′-OHterminus of the second strand for initiation, or incorporating apromoter sequence (for example, flanking the target binding arm).

In one embodiment, a method of template-directed nucleic acid synthesisrequiring a primer is performed by (i) binding a target nucleic acid toa split G-quadruplex associated to a capture tag, and binding thecapture tag to a solid surface; and (ii) initiating nucleic acidsynthesis using the 3′-OH terminus of the second strand of the splitG-quadruplex. In one preferred embodiment, the 3′-OH terminus of thefirst strand of the split G-quadruplex is modified (ex. aminated) toprevent synthesis from the first strand. In a second preferredembodiment, the temperatures for target nucleic acid hybridization andenzymatic extension are below the melting temperature of theG-quadruplex. In a second embodiment, a method of template-directednucleic acid synthesis requiring a promoter is performed by (i) bindinga target nucleic acid to a split G-quadruplex associated to a capturetag and a promoter sequence, and binding the capture tag to a solidsurface; and (ii) initiating nucleic acid synthesis using the promotersequence associated to the split G-quadruplex. In a preferredembodiment, the promoter sequence is incorporated in a region flankingthe target-binding arm. In another preferred embodiment, the promotersequence is double-stranded nucleic acid, for example, formed byhybridizing a complementary sequence incorporated in the same or adifferent strand of the split G-quadruplex, or by hybridizing acomplementary sequence incorporated in a nucleic acid fragment.

Template-directed nucleic acid synthesis of the target nucleic acid canbe used to strengthen the binding between the split G-quadruplex and thetarget nucleic acid. For example, if the nucleic acid synthesis isinitiated at the 3′-OH terminus of the split G-quadruplex and isextended along the length of the target nucleic acid (downstream of thesplit G-quadruplex), it creates a complementary nucleic acid that isconnected to the split G-quadruplex and bound to the target nucleicacid. Depending on the length of the target nucleic acid that isdownstream of the split G-quadruplex, the newly synthesizedcomplementary fragment can be large, and can be used to strengthen thebinding the split G-quadruplex and the target nucleic acid. In oneembodiment, the binding of a split G-quadruplex bound to a targetnucleic acid is strengthen by (i) binding the split G-quadruplex to atarget nucleic acid and (ii) performing template-directed nucleic acidsynthesis on the target nucleic acid. In another embodiment, afunctional tag can be associated to a split G-quadruplex by (i) bindingthe split G-quadruplex to a target nucleic acid, and (ii) performingtemplate-directed nucleic acid synthesis on the target nucleic acidutilizing nucleotides bound to functional tags.

Use of split G-quadruplexes for template-directed nucleic acid synthesishas two key advantages over single-stranded primers (with sequence equalto the target-binding arms): (i) split G-quadruplexes can be designed tohybridize at much different temperatures in comparision to singlestranded primers, for example by decreasing the length of one arm andincreasing the length of the other arm, while maintaining the sameoverall sequence; and (ii) split G-quadruplexes are more specific thansingle-stranded primers because the individual target binding arms canbe shorter than primers, and thus more sensitive to nucleotidesubstitutions, especially if one arm is made short in length.

Examples of commonly used solid surfaces for binding of nucleic acidsinclude glass slides, silicon chips, micro-beads, micro-spheres, andsedimentable and ferromagnetic substances, such as agarose resin andiron beads. Examples of tags (and corresponding reactive groups orcoatings on solid surfaces) include amino modifications (and epoxysilane or isothiocynanate coated surfaces), thiol modifications (andmercaptosilanized surfaces), hydrazide modifications (and aldehyde orepoxide), biotin (and immobilized streptavidin), cholesterol-TEG (andimmobilized anti-cholesterol antibodies), and digoxigenin NHS Ester (andimmobilized anti-digoxigenin antibodies). Some tags bind directly to thereactive groups on the solid surface (ex. biotin and streptavidin), andother tags require a chemical reaction with secondary chemicals forattachment. Examples of micro-spheres include polystyrene micro-spheres,magnetic micro-spheres, and silica micro-spheres.

Split G-quadruplexes are advantageous for the capture of target nucleicacids, including (1) split G-quadruplexes can selectively capture atarget nucleic acid based on sequence, with SNP specificity; (2) splitG-quadruplexes can be readily used for template-directed nucleic acidsynthesis of the target nucleic acid that has been captured, (3) splitG-quadruplex can be easily denatured (ex. thermally, chemically) ordigested (ex. at a engineered restriction site by a restriction enzyme)to liberate the target nucleic acid that has been captured, and (4)split G-quadruplexes can be simpler to modify than nucleic acids (ex.long synthetic nucleic acids, and especially nucleic acids isolated frombiological sources). Further, the efficiency of capture can be easilymonitored, for example, by (i) capturing a target nucleic acid onto asolid surface with a split G-quadruplex associated to a detection tag(or detectable capture tag) on one strand and a capture tag on the otherstrand (FIG. 1 Embodiment 4), (ii) removing (ex. by washing) the unboundstrand with the detection tag, and (iii) monitoring the detection tag ofthe bound second strand by methods known in the art. Alternatively, onecan (i) capture a target nucleic acid onto a solid surface with a splitG-quadruplex with a detection tag on one stand (or both strands), and(ii) monitor the assembled split G-quadruplexes by monitoring itscatalytic activity.

In one embodiment, the capture tag is associated to a strand of theG-quadrupex via a linker or spacer—for example, Phosphoramidite C3,Hexanediol, and 1′,2′-Dideoxyribose, and preferentially TriethyleneGlycol or Hexa-Ethyleneglycol. In cases where there is poor bindingbetween the capture tag and the solid surface—ex. due to sterichinderance, incompatible surface charge, incompatible surfacehydrophobicity/hydrophilicity—the linker of the capture tag can be madelonger. For example, the linker can be made longer adding additionallinker molecules to the first linker (ex. (HEG)₅, which is 5Hexa-Ethyleneglycol linkers). Or alternatively, the linker can be madelonger by adding additional nucleotides (ex. dT₁₀, which is 10deoxythymines) between the capture tag and the other parts of the splitG-quadruplex (ex. the target binding arm and the partial G-quadruplexsequence).

In the present invention, split G-quadruplexes and capture nucleic acidscan be used together for the capture of target nucleic acids to solidsurfaces. In a preferred embodiment, the split G-quadruplex has capturearms capable of binding the capture nucleic acid, and the capturenucleic acid has a capture tag capable of binding the solid surface. Inone embodiment, a split G-quadruplex associated to a capture tag can beused to bind a target nucleic acid to a solid surface by (i) associatinga capture tag to a capture nucleic acid, (ii) binding said capture tagto a solid surface, (iii) binding the capture arm(s) of a splitG-quadrupex to said capture nucleic acid, and (iv) binding a targetnucleic acid to the target binding arms of said split G-quadruplex. In asecond embodiment, a split G-quadruplex associated to a capture tag canbe used to bind a target nucleic acid to a solid surface by (i)associating a capture tag to a capture nucleic acid, (ii) binding saidcapture tag to a solid surface, (iii) binding the target binding arms ofa split G-quadruplex to a target nucleic acid, and (iv) binding thecapture arms of said split G-quadruplex to said capture nucleic acid. Anillustration of a target nucleic acid associated to a capture tag—i.e.the capture arms of a split G-quadruplex—and a solid surface using saidmethods, is shown in FIG. 1 Embodiment 5. Said target nucleic acid canbe detected using a second G-quadruplex associated with a detection tag(or a capture tag capable of binding detectable capture targets)—asshown in FIG. 1 Embodiment 6.

The present invention features a kit and an apparatus for using splitG-quadruplexes with functional tags, for example, to detect or captureor target or crosslink target nucleic acids. The kit or apparatus can bepoint-of-care (POC). In one embodiment, the kit or apparatus includes asplit G-quadruplex associated with a functional tag. In anotherembodiment, the kit or apparatus includes a split G-quadruplexassociated with a capture tag, and a solid surface (capable of bindingsaid capture tag). And in another embodiment, the kit or apparatusincludes a split G-quadruplex associated with a capture tag (capable ofbinding a capture nucleic acid), a capture nucleic acid with a capturetag (capable of binding a solid surface), and a solid surface.

EXAMPLES

Other features and advantages of the invention will be apparent from thefollowing examples of the embodiments and from the claims.

Example 1 Strong Binding by Split G-Quadruplexes

To observe the binding of split G-quadruplexes on target nucleic acids(in absence of G-quadruplex catalytic activity), a target nucleic acidwith a capture tag was bound to a solid surface, washed, bound with asplit G-quadruplex, washed repeatedly, and then the bound splitG-quadruplex was detected. The utilized target nucleic acid had acapture tag and sequence 5′-/5Biosg/NN NNN NNN NNN NNN NNN NNN NNN NNNNNN NNN NNN NNN NNN NNN NNN NNN NAA CCA TTT GGG TGT CCT GAT-3′ (SEQ IDNO: 1). The utilized split G-quadruplex, specific for said targetnucleic acid, comprised two oligonucleotide strands, with the firststrand of sequence 5′-AT CAG GAC AC/iSp9/GGG TTG GG-3′ (SEQ ID NO: 2)and the second strand of sequence 5′-GGG TAG GG/iSp9/CCA AAT GG-3′ (SEQID NO: 3). Oligonucleotides were custom-made by IDT (Coralville, Iowa).Other reagents, unless otherwise indicated, were purchased fromSigma-Aldrich (St. Louis, Mo.).

Target nucleic acid (100 pm) was first bound to a solid surface—PierceStreptavidin Coated High Capacity Plates (Thermo Fischer Scientific,Waltham, Mass.)—for 1 hour at 37° C. in Binding Buffer (50 mM HEPES (pH7.4), 20 mM KCl, 50 mM NaCl, 0.02% Triton X-100 (0.02% v/v), 50 mMMgCl₂). The plates were washed in Wash Buffer (TBS, 0.1% BSA, 0.05%Tween-20), and then bound with 100 pm split G-quadruplex for 30 minutesat 25° C. in Binding Buffer. Plates were either (i) washed one time or(ii) washed one time, rested for 30 minutes, and washed again one time.To observe binding, the catalytic activity of the bound splitG-quadruplex was triggered in Binding Buffer supplemented with 125 mMhemin, 1 mM H₂O₂, 1 mM ABTS; and the 420 nm absorbance of the resultingproduct was measured on a SpectraMax Plus 384 spectrophotometer(Molecular Devices, Sunnyvale, Calif.). The results are presented inTable 1, and demonstrate that split G-quadruplexes bind target nucleicacids in absence of catalytic activity—and the binding is stable afterone wash or two washes with an added rest step.

TABLE 1 Effect of Washes on Binding of Split G-Quadruplex to TargetNucleic Acids A420 1 Wash 0.66 1 Wash + 30′ Rest + 1 Wash 0.65 No Target(70329) 0.10

Cooperative Binding by Split G-Quadruplexes

The strong binding suggests that the two strands of the splitG-quadruplex (on binding the target nucleic acid) bind one another viaformation of the G-quadruplex, and this binding further stabilizes eachstrand on the target nucleic acid. To determine if the binding of thetwo strands of the split G-quadruplex on the target is cooperative, atarget nucleic acid with a capture tag (SEQ ID NO: 1) (100 pm) was boundto a solid surface for 30 minutes at 25° C., washed, and then bound for30 minutes at 25° C. with either (i) 100 pm of the first strand of asplit G-quadruplex (SEQ ID NO: 2) (herein called well 1) or (ii) 100 pmof the second strand of a split G-quadruplex (SEQ ID NO: 3) (hereincalled well 2) or (iii) both strands of a split G-quadruplex (SEQ ID NO:2 and SEQ ID NO: 3) (herein called well 3). Afterwards, the supernatantof well 1 (containing unbound SEQ ID NO: 2) and well 2 (containingunbound SEQ ID NO: 3) was transferred to well A containing 100pm oftarget nucleic acid of sequence 5′-AA CCA TTT GGG TGT CCT GAT-3′ (SEQ IDNO: 4); and the supernatant of well 3 (containing unbound SEQ ID NO: 2and SEQ ID NO:3) was transferred to well b containing 100 pm of targetnucleic acid of sequence SED ID NO: 4. To observe binding, the catalyticactivity of unbound split G-quadruplexes was triggered, and the 420 nmabsorbance of the resulting product was measured on a spectrophotometer.The results are presented in Table 2 and indicate cooperativebinding—that is, more strands are retained on target nucleic acids whenthe strands are bound jointly (because of cooperative binding) (ex. well3) and not separately (ex. well 1 and well 2).

TABLE 2 More Retention of Split G-Quadruplex Strands Bound Jointly ThanSeparately A420 Well A (bound separately) 0.56 Well B (bound jointly)0.32 No Target (61208) 0.03

Example 2 Detection of Target Nucleic Acid by Split G-Quadruplex withCapture Tag

The strong binding and the cooperative binding observed in Example 1—inabsence of G-quadruplex catalytic activity—indicates that splitG-quadruplexes stably bind target nucleic acids, and accordingly, theycan also be used to associate other molecules such as functional tags(bound or incorporated into the split G-quadruplex) to target nucleicacids. To demonstrate that a capture tag of a split G-quadruplex can beassociated to a target nucleic acid—and moreover, a detectable capturetarget can be bound to the capture tag for detection of the targetnucleic acid—a target nucleic acid with a capture tag (SEQ ID NO: 1) wasbound to a solid surface, washed, bound with a split G-quadruplex with aDIG capture tag (SEQ ID NO: 2 and sequence 5′-GGG TAG GG/iSp9/CCA AATGG/3DiG_N/-3′ (SEQ ID NO: 5)), washed, bound for 30 minutes at 25° C.with rabbit anti-DIG antibody (Thermo Fischer Scientific), washed, boundfor 30 minutes at 25° C. with anti-rabbit antibody conjugated to HRP(Sigma-Aldrich), washed, and then the catalytic activity of the HRP wastriggered in Binding Buffer supplemented with 1 mM H₂O₂ and 1 mM ABTS,and the 420 nm absorbance of the resulting product was measured on aspectrophotometer. (The method closely resembles FIG. 1 Embodiment 2,except it uses two antibodies instead of one for detection). Othersolutions and methods are the same as Example 1 unless otherwiseindicated. The results are presented in Table 3, and demonstrate the useof split G-quadruplexes to (i) associate functional tags, and inparticular capture tags, to target nucleic acids, (ii) associate capturetargets to target nucleic acids, and (iii) associate detectablemolecules to target nucleic acids via binding to split G-quadruplexcapture tags.

TABLE 3 Detection of Target Nucleic Acid using Split G-Quadruplex withCapture Tag A420 Antibody + Target Nucleic Acid 1.35 Antibody Alone(70703) 0.15

Example 3 Detection of Target Nucleic Acid by Split G-Quadruplex withCapture Arms

Split G-quadruplexes can be designed with additional target binding arms(herein called capture arms), which are capable of binding additionaltarget nucleic acids (herein called capture nucleic acids). In thefollowing example, a split G-quadruplex with two target arms and twocapture arms was designed [5′-AT CAG GAC AC/iSp9/GGG TTG GG/iSp9/ATT AAGTGT-3′ (SEQ ID NO: 6) and 5′-GGC CAG TTT CAT TTG AGC/iSp9/GGG TAGGG/iSp9/CCA AAT GG-3′ (SEQ ID NO: 7)], which was capable of binding twocapture nucleic acids, that is, two strands of a second splitG-quadruplex of sequence [5′-ACA CTT AAT/iSp9/GGG TTG GG-3′ (SEQ ID NO:8) and 5′-GGG TAG GG/iSp9/GCT CAA ATG AAA CTG CCC-3′ (SEQ ID NO: 9)]. Todemonstrate the methodology, a target nucleic acid with a capture tag(SEQ ID NO: 1) was bound to a solid surface, washed, bound with a splitG-quadruplex with two target arms and two capture arms (SEQ ID NO: 6 andSEQ ID NO: 7), washed, and bound with two capture nucleic acids thatwere also strands of a second split G-quadruplex (SEQ ID NO: 8 and SEQID NO: 9), which is capable of assembling into a functional G-quadruplex(see FIG. 1 Embodiment 2).

TABLE 4 Detection of a Capture Nucleic Acid - a 2^(nd) Split GQuadruplex A420 1^(st) + 2^(nd) Split G-Quadruplexes 0.86 1^(st) SplitG-Quadruplex Alone 0.26 No Target (60310) 0.17

To observe binding of the captured nucleic acids, the catalytic activityof both split G-quadruplexes was triggered, and the 420 nm absorbance ofthe resulting product was measured on a spectrophotometer. Othersolutions and methods are the same as Example 1 unless otherwiseindicated. The results are presented in Table 4 above, and demonstratethe use of capture arms on split G-quadruplexes to capture other nucleicacids (that is, capture nucleic acids), and the use of said capturenucleic acids for the detection of target nucleic acids. This approachcan be used with other types of detectable nucleic acids in place of thesecond G-quadrupex, for example, nucleic acids with detection tags, andnucleic acids with capture tags capable of binding detectable molecules.The approach can also be used with capture nucleic acids associated withother types of functional tags (ex. capture tags, target tags,crosslinked tags, etc.), which can be used for the capture, targeting,or crosslinking of target nucleic acids.

Example 4 Capture of Target Nucleic Acid by Split G-Quadruplex withCapture Tag

Several types of capture tags are capable of binding solid surfaces.These capture tags, associated to split G-quadruplexes, can be used tobind split G-quadruplexes to solid surfaces. Moreover, these splitG-quadruplexes, capable of binding target nucleic acids, can be used tobind (capture) target nucleic acids to solid surfaces. To demonstratethat split G-quadrplexes can be used to capture target nucleic acidsonto solid surfaces, one strand of a split G-quadruplex with a biotincapture tag [5′-/SBiosG/AT CAG GAC AC/iSp9/GGG TTG GG-3′ (SEQ ID NO: 10)] was bound to a solid surface, washed, bound with a target nucleic acid(SEQ ID NO: 4) and the second strand of the G-quadruplex with a DIGcapture tag (SEQ ID NO: 5), washed, bound with rabbit anti-DIG antibody,washed, bound with anti-rabbit antibody conjugated to HRP, washed, andthen the catalytic activity of the HRP was triggered in Binding Buffersupplemented with 1 mM H₂O₂ and 1 mM ABTS, and the 420 nm absorbance ofthe resulting product was measured on a spectrophotometer. The method issimilar to FIG. 1 Embodiment 4, with the drawn detection tag replacedwith a capture tag, which capable of binding an antibody (which iscapable of binding a 2^(nd) antibody). Other solutions and methods arethe same as Example 2 unless otherwise indicated. The results arepresented in Table 5, and demonstrate the use of split G-quadruplexes tocapture target nucleic acids onto solid surfaces.

TABLE 5 Capture of a Target Nucleic Acid by Split G Quadruplex withCapture Tag A420 Antibody + Target Nucleic Acid 0.50 Antibody Alone(70412) 0.20

Example 5 Capture of Assembled Target Nucleic Acid+Split G-Quadruplex

A second approach to capture target nucleic acids onto solid surfacesbinds split G-quadruplexes (with capture tags) to target nucleic acids,and then binds the (capture tag with the) assembled complex to a solidsurface. This approach can be optimized by using a smaller amount of thesplit G-quadruplex strand with the capture tag relative to its otherstrand. To demonstrate this approach, a split G-quadruplex with Biotinand DIG capture tags (25 pm of SEQ ID NO: 10 and 100 pm of SEQ ID NO: 5)was mixed for 60 minutes with 100 pm of target nucleic acid, and thenbound to a solid surface (coated with avidin). The bound, assembledcomplexes were then washed, bound with rabbit anti-DIG antibody, washed,bound with anti-rabbit antibody conjugated to HRP, washed, and then thecatalytic activity of the HRP was triggered in Binding Buffersupplemented with 1 mM H₂O₂ and 1 mM ABTS, and the 420 nm absorbance ofthe resulting product was measured on a spectrophotometer. Othersolutions and methods are the same as Example 4 unless otherwiseindicated. The results are presented in Table 6, and demonstrate asecond approach to capture target nucleic acids onto solid surfacesusing split G-quadruplexes.

TABLE 6 Capture of Assembled Target Nucleic Acid/Split G Quadruplex withCapture Tag A420 Assembled Target/Quadruplex 1.00 No Target (70320) 0.20

What is claimed is:
 1. A method of associating a tag to a nucleic acid,comprising: associating the tag to a split G-quadruplex, and binding thesplit G-quadruplex to the nucleic acid.
 2. The method of claim 1,further comprising performing template-directed nucleic acid synthesisof the nucleic acid.
 3. The method of claim 1, wherein the associatingstep is accomplished by binding or incorporating the tag into one strandof the split G-quadruplex.
 4. The method of claim 1, wherein theassociating step is accomplished by binding or incorporating the taginto two strands of the split G-quadruplex.
 5. The method of claim 1,wherein the tag is an attachment chemistry, a fluorophore, a detectableenzyme, a detectable particle, or a nucleotide analog.
 6. The method ofclaim 1, wherein the tag is a detection tag, a capture tag, a targetingtag, or a crosslinking tag.
 7. The method of claim 6, wherein thecapture tag is a capture arm.
 8. The method of claim 1, wherein the tagis associated to the split G-quadruplex by template-directed nucleicacid synthesis.
 9. A method of detecting a nucleic acid, comprising:associating a detection tag or capture tag to a split G-quadruplex,binding the split G-quadruplex to the nucleic acid, and detecting thedetection tag or capture tag.
 10. The method of claim 9, wherein thecapture tag is detected by binding a detectable capture target to thecapture tag, and detecting the detectable capture target.
 11. The methodof claim 9, further comprising binding the nucleic acid to a solidsurface.
 12. The method of claim 11, comprising: associating a detectiontag or capture tag to a split G-quadruplex, binding the capture tag ifpresent to a detectable capture target, binding the split G-quadruplexto a nucleic acid, binding the nucleic acid to a solid surface, washingthe solid surface to remove unbound molecules, and detecting the bounddetection tag or detectable capture target.
 13. The method of claim 9,wherein the detection tag or capture tag is associated to the splitG-quadruplex by template-directed nucleic acid synthesis.
 14. A methodof capturing a nucleic acid, comprising: associating a capture tag to asplit G-quadruplex, binding the split G-quadruplex to the nucleic acid,and binding the capture tag to a capture target.
 15. The method of claim14, wherein the capture target is a nucleic acid, a molecule that can bedetected, a molecule that can bind other molecules, or a solid surface.16. The method of claim 15, wherein the capture target is a solidsurface.
 17. The method of claim 15, wherein the capture tag is acapture arm, and the capture target is a capture nucleic acid.
 18. Themethod of claim 17, further comprising binding the capture nucleic acidto a solid surface.
 19. The method of claim 14, wherein the splitG-quadruplex has a detection tag or a second capture tag capable ofbinding a detectable capture target.
 20. The method of claim 14, whereinthe capture tag is associated to the split G-quadruplex bytemplate-directed nucleic acid synthesis.